scholarly journals Optimization of an Efficient Protein Extraction Protocol Compatible with Two-Dimensional Electrophoresis and Mass Spectrometry from Recalcitrant Phenolic Rich Roots of Chickpea (Cicer arietinum L.)

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Moniya Chatterjee ◽  
Sumanti Gupta ◽  
Anirban Bhar ◽  
Sampa Das
Keyword(s):  
Mass Spectrometry ◽  
Protein Extraction ◽  
Sodium Dodecyl ◽  
Good Resolution ◽  
Two Dimensional ◽  
Two Dimensional Electrophoresis ◽  
Extraction Protocol ◽  
Phenol Extraction ◽  
Spot Number ◽  
Dimensional Electrophoresis

Two-dimensional electrophoresis and mass spectrometry are undoubtedly two essential tools popularly used in proteomic analyses. Utilization of these techniques however largely depends on efficient and optimized sample preparation, regarded as one of the most crucial steps for recovering maximum amount of reliable information. The present study highlights the optimization of an effective and efficient protocol, capable of extraction of root proteins from recalcitrant phenolic rich tissues of chickpea. The widely applicable TCA-acetone and phenol-based methods have been comparatively evaluated, amongst which the latter appeared to be better suited for the sample. The phenol extraction-based method further complemented with sodium dodecyl sulphate (SDS) and pulsatory treatments proved to be the most suitable method represented by greatest spot number, good resolution, and spot intensities. All the randomly selected spots showed successful identification when subjected to further downstream MALDI-TOF and MS/MS analyses. Hence, the information obtained collectively proposes the present protein extraction protocol to be an effective one that could be applicable for recalcitrant leguminous root samples.

scholarly journals Evaluation of six different protocols for protein extraction from rice young panicles by two-dimensional electrophoresis

2020 ◽  
Author(s):  
Xiaoping Huang ◽  
Hui-wen Zhou
Keyword(s):  
Protein Concentration ◽  
Protein Extraction ◽  
Image Resolution ◽  
Plant Tissues ◽  
Two Dimensional ◽  
Protein Patterns ◽  
Molecular Weight Range ◽  
Two Dimensional Electrophoresis ◽  
Phenol Extraction ◽  
Dimensional Electrophoresis

Abstract Background: For most reported proteomics approaches, protein extraction are of crucial importance for optimal results. However, extraction of protein from plant tissues still exist great challenges due to low protein content and many secondary metabolites that prominently interfering with isoelectric focusing. Up to now, no attempts are focused on comparison of protein extraction from rice young panicles.Methods: To establish a higher efficiency protein extraction protocol suited for two-dimensional electrophoresis in rice young panicles, six protocols for protein preparation were evaluated in terms of protein concentration, the molecular weight range of protein, gel image resolution, the number of protein spots: 1) Phenol extraction; 2) Mg/Nonidet P-40 (NP-40) extraction; 3) Tris-Base/acetone extraction; 4) SDS extraction; 5) trichloroacetic acid (TCA)/acetone/phenol extraction; 6) TCA/acetone precipitation.Results: The result explicitly demonstrated that TCA/acetone/phenol method provided a high-enhanced protein extraction efficacy from rice young panicles than other protocols in terms of the protein concentration (9.79±0.23 SD), the most comprehensive proteins (10 KDa to 150 KDa), the maximum number of protein spots (450±53 SD), the greater gel image resolution and spot abundance. In addition, these methods also generated remarkably differential 2-DE protein patterns. Twenty-nine of 30 visible differentially extracted proteins were identified by MS analysis and were divided into eight categories. Prediction for protein subcellular localization and grand average of hydropathy (GRAVY) analysis showed that certain special proteins respectively necessitate different extraction methods due to different physicochemical properties of each protocol.Conclusions: Overall, this paper will facilitate to provide a cornerstone of comparative proteomic analysis from rice young panicles, including other complicated plant tissues.

scholarly journals Application of two-dimensional electrophoresis and mass spectrometry to screen endometriosis-related proteins

2014 ◽  
Vol 10 (1) ◽  
pp. 95-100
Author(s):  
HANIKEZI TUERXUN ◽  
YANMEI ZHANG ◽  
FEI JI ◽  
AIXINGZI AILI ◽  
XINHUA YANG ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Two Dimensional ◽  
Two Dimensional Electrophoresis ◽  
Related Proteins ◽  
Dimensional Electrophoresis

Two-dimensional electrophoresis of human placental mitochondria and protein identification by mass spectrometry: Toward a human mitochondrial proteome

1998 ◽  
Vol 19 (6) ◽  
pp. 1006-1014 ◽  
Author(s):  
Thierry Rabilloud ◽  
Sylvie Kieffer ◽  
Vincent Procaccio ◽  
Mathilde Louwagie ◽  
Paul L. Courchesne ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Identification ◽  
Two Dimensional ◽  
Mitochondrial Proteome ◽  
Two Dimensional Electrophoresis ◽  
Dimensional Electrophoresis

scholarly journals Red cell proteins. I. Two-dimensional mapping of human erythrocyte lysate proteins

Blood ◽  
1979 ◽  
Vol 53 (6) ◽  
pp. 1121-1132 ◽  
Author(s):  
JJ Edwards ◽  
NG Anderson ◽  
SL Nance ◽  
NL Anderson
Keyword(s):  
Human Erythrocyte ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Cell Protein ◽  
Red Cell ◽  
Two Dimensional ◽  
Two Dimensional Electrophoresis ◽  
Mapping Techniques ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Dimensional Electrophoresis

Abstract Human erythrocyte lysate proteins were resolved into over 250 discrete spots by two-dimensional electrophoresis using isoelectric focusing in the first dimension and electrophoresis in the presence of sodium dodecyl sulfate, (SDS) in the second. The overwhelming excess of hemoglobin has made such analyses difficult in the past. However, with the ISO-DALT two-dimensional electrophoresis system, large numbers of red cell proteins can be mapped in the presence of hemoglobin. When hemoglobin and several other major proteins are removed by adsorption to DEAE-cellulose, additional minor components are seen, giving a total of over 275. With the use of purified preparations, the map positions of five cell enzymes or their subunits were determined: pyruvate kinase, catalase, glucose-6-phosphate dehydrogenase, hypoxanthine phosphoribosyltransferase, and carbonic anhydrase. The mapping techniques described complement and extend those traditionally used to find human red cell protein variants.

Evaluation of lipoproteins and apolipoproteins in serum of a Tangier patient by micro-scale two-dimensional electrophoresis.

1987 ◽  
Vol 33 (4) ◽  
pp. 468-472 ◽  
Author(s):  
T Manabe ◽  
S Visvikis ◽  
M F Dumon ◽  
M Clerc ◽  
G Siest
Keyword(s):  
Molecular Mass ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Disease Patient ◽  
High Density Lipoproteins ◽  
Two Dimensional ◽  
High Concentration ◽  
Two Dimensional Electrophoresis ◽  
Micro Scale ◽  
Dimensional Electrophoresis

Abstract We examined lipoproteins and apolipoproteins in serum of a Tangier-disease patient. We used three different techniques of micro-scale two-dimensional electrophoresis: (a) no denaturants; (b) with sodium dodecyl sulfate (SDS) used only in the slab gel electrophoresis; (c) and with urea and a detergent used in isoelectric focusing and with SDS in slab gel electrophoresis. By technique a, an extremely low concentration of high-density lipoproteins (HDL) in the Tangier serum was seen, and lipoproteins that cannot form HDL complexes were detected as multiple spots in the acidic (pl 4 approximately 5) and relatively low apparent molecular mass (20,000 approximately 80,000) region. By technique b, Tangier low-molecular-mass lipoproteins were dissociated into their constituent apolipoproteins, and we observed a higher proportion of apoC-III, together with lower proportions of apoA-I and apoA-II, than in the normal HDL fraction. Technique c showed the total content of apolipoproteins in the whole Tangier serum, as several workers have reported. The presence of low-molecular-mass lipoproteins and a high concentration of apoC-III in this lipoprotein fraction characterized the Tangier serum.

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